The general public is highly interested in health and hygiene as the level of income increases. However, it is not easy for them to personally detect and quantitatively evaluate microorganisms that are invisible to the naked eye.
Various microorganisms inhabit daily items such as hair combs, cell phones, desks, clothes with which a human being uses, or spaces such as bathrooms, bedrooms, or those microorganisms are present even in the air. Such microorganisms are likely to contain opportunistic pathogen and pathogenic bacteria, and the like.
Traditional methods for measuring the amount of microorganisms include a standard plating method in which a sample taken from an environment is serially diluted and smeared on a medium capable of culturing microorganisms, and 2 or 3 days later, the number of colonies produced from the medium is calculated to estimate the amount of microorganisms. Such traditional methods have disadvantages that they require specialized experimental tools and skilled technicians as well as a very long time, thereby making it difficult for the general public to quickly measure living microorganisms.
Recently, several methods have been developed to solve the problems associated with conventional methods. Such methods indirectly measure the amount of microorganisms by measuring the amount of various constituent substances which are constitutively present in cells.
One representative method is to perform a measurement based on the amount of ATP. ATP is a useful substance for measuring the amount of cells in a sample, since it is used as a major source of bioenergy in cells and is a constituent which all organisms have in common. Due to these advantages, an ATP-based measurement method is widely used for quantitative measurement of microorganisms. In order to measure ATP, a luciferase enzyme is generally used to induce luminescence reaction and quantification is made through intensity of light. In this case, luciferin is used as a substrate.
However, the ATP-based method has disadvantages that ATP in a reaction sample is rapidly depleted and thus signals do not last for a long time, and production costs of the enzyme and substrate to be added are high. In addition, there is a problem that it has limited storability due to the reason that the constituent used for measurement is an enzyme. There is another problem in the method of quantifying microbial cells through such ATP measurement. That is, microorganisms that are dead or have a very low viability are most likely to be measured as well, since the function of ATP is maintained even if cells are dead.
Another method is to use ligases. This method is initiated by adding specific DNA fragments into a sample. Then, ligases in cells recognize the DNA fragments and ligate two fragments through polymerization reaction. It is known that when real-time PCR analysis is performed for the thus ligated fragments using specific primers, it is possible to evaluate the amount of microorganisms at even 103 cells.
However, as in the case of the measurement methods performed through luciferase, this method has difficulty with respect to storage since it uses ligase which is an enzyme. Also, in order to identify new nucleic acid molecules produced by the reaction of ligases with DNA strands, RT-PCR and PCR, gel electrophoresis procedure are needed, thereby requiring expensive equipment and skilled technicians as well as a very long time for analysis. Further, since ATP molecules are also present on dead cells as in the measurement method using luciferase, it has a disadvantage that it is not possible to measure only living microorganisms.
Accordingly, in order to solve such problems, it is desirable to estimate the detection and/or quantity of microorganisms by measuring the amount and activity of an enzyme that is constitutively present in cells.
(Prior art Document) Patent Application No. KR1020100081068